Analytical HPLC to Preparative HPLC
Scale-Up Techniques Using a Natural Product Extract
Introduction
Chromatographic separation methods can be developed on any scale. To minimize consumption of sample and solvents there is a benefit in developing separation methods on a small scale and transferring them to a larger scale. Taking into account important parameters and applying appropriate scaling factors in a logical manner enables users to scale up from analytical chromatography to preparative separations easily and successfully. In this application note an analytical scale separation of Kudzu root extract is used to demonstrate the calculations and techniques used to move from a 4.6 mm i.d. analytical column separation through 10, 19, and 30 mm i.d. preparatory column separations.
Experimental Conditions
Separations, at all scales, were carried out using the Waters® AutoPurifcation System consisting of the following components:
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2545 Binary Gradient Module
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2998 Photo Diode Array
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3100 Mass Detector
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2767 Sample Manager
- System Fluidics Organizer
Discussion
A systematic approach to scale up will provide the best possible result. The ultimate goals are to maintain chromatographic resolution between key components and enable users to better predict chromatographic performance between analytical and preparative chromatography.
Columns of the same particle size, length, and chemistry will provide similar resolution of critical pairs at all separation scales. Waters offers a wide range of column chemistry choices available in analytical and preparative scale dimensions.
To demonstrate the technique, an analytical scale separation was developed on a 4.6 X 50 mm Waters SunFire C18, 5 μm column. This separation was scaled to three different preparative dimension columns (10.0, 19.0, and 30.0 mm i.d.). The scaled flow rates, injection volumes, and gradient tables were all calculated using the Waters OBD Prep Calculator.
Results
Regardless of the scale, the chromatography is very similar (figure 1). When compared to the original 4.6 mm i.d. scale, resolutions and retention times are very similar across all scales. This simple experiment demonstrates that a systematic approach to scale up meets the goal of maintaining chromatographic resolution between key components and enables users to better predict chromatographic performance between analytical and preparative chromatography. This exercise also demonstrates the unique capability of the Waters AutoPurification system which allows users to perform both analytical and preparatory chromatography on the same system with no performance compromise.
Conclusions
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Analytical chromatography can be successfully scaled to preparatory chromatography easily by using a systematic approach.
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The Waters Prep OBD Calculator aids in the scaling calculations.
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Using the Waters AutoPurification System, separation methods developed on an analytical scale can be transferred to preparatory scale on the same system.
- Developing methods on the analytical scale and transferring them to preparatory scale reduces solvent and sample consumption while reducing waste disposal cost compared to developing separation methods only at the preparatory scale.
Reference
Analytical HPLC to Preparative HPLC: Scale-up techniques using a natural product extract. June 2009 72000312EN
Source: Waters Corporation
